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. 2016 Jan;25(1):54-64.
doi: 10.1097/CEJ.0000000000000141.

Synergic chemoprevention with dietary carbohydrate restriction and supplementation of AMPK-activating phytochemicals: the role of SIRT1

Jong Doo Lee  1 , Min-Ah Choi, Simon Weonsang Ro, Woo Ick Yang, Arthur E H Cho, Hye-Lim Ju, Sinhwa Baek, Sook In Chung, Won Jun Kang, Mijin Yun, Jeon Han Park
Affiliations

Synergic chemoprevention with dietary carbohydrate restriction and supplementation of AMPK-activating phytochemicals: the role of SIRT1

Jong Doo Lee et al. Eur J Cancer Prev. 2016 Jan.

Abstract

Calorie restriction or a low-carbohydrate diet (LCD) can increase life span in normal cells while inhibiting carcinogenesis. Various phytochemicals also have calorie restriction-mimetic anticancer properties. We investigated whether an isocaloric carbohydrate-restriction diet and AMP-activated protein kinase (AMPK)-activating phytochemicals induce synergic tumor suppression. We used a mixture of AMPK-activating phytochemical extracts including curcumin, quercetin, catechins, and resveratrol. Survival analysis was carried out in a B16F10 melanoma model fed a control diet (62.14% kcal carbohydrate, 24.65% kcal protein and 13.2% kcal fat), a control diet with multiple phytochemicals (MP), LCD (16.5, 55.2, and 28.3% kcal, respectively), LCD with multiple phytochemicals (LCDmp), a moderate-carbohydrate diet (MCD, 31.9, 62.4, and 5.7% kcal, respectively), or MCD with phytochemicals (MCDmp). Compared with the control group, MP, LCD, or MCD intervention did not produce survival benefit, but LCDmp (22.80±1.58 vs. 28.00±1.64 days, P=0.040) and MCDmp (23.80±1.08 vs. 30.13±2.29 days, P=0.008) increased the median survival time significantly. Suppression of the IGF-1R/PI3K/Akt/mTOR signaling, activation of the AMPK/SIRT1/LKB1pathway, and NF-κB suppression were the critical tumor-suppression mechanisms. In addition, SIRT1 suppressed proliferation of the B16F10 and A375SM cells under a low-glucose condition. Alterations in histone methylation within Pten and FoxO3a were observed after the MCDmp intervention. In the transgenic liver cancer model developed by hydrodynamic transfection of the HrasG12V and shp53, MCDmp and LCDmp interventions induced significant cancer-prevention effects. Microarray analysis showed that PPARα increased with decreased IL-6 and NF-κB within the hepatocytes after an MCDmp intervention. In conclusion, an isocaloric carbohydrate-restriction diet and natural AMPK-activating agents induce synergistic anticancer effects. SIRT1 acts as a tumor suppressor under a low-glucose condition.

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Figures

Fig. 1
Fig. 1
Dietary effects on survival. (a) Kaplan–Meier survival analysis showed that AMPK-activating phytochemicals alone do not produce survival benefit in a melanoma model. The median survival time was not significantly increased after the multiple phytochemical (MP) intervention. (b) Survival benefit was not observed after the low-carbohydrate diet (LCD) intervention either. However, LCD supplemented with a mixture of phytochemicals (LCDmp) significantly increased the median survival time. The median survival time of the control, LCD, and LCDmp groups was 22.80±1.58, 24.69±1.42, and 28.00±1.64 days after inoculation of melanoma cells, respectively. (c) The median survival time of the MCDmp group was also significantly increased compared with that of the MCD and control groups (30.13±2.29, 25.40±1.24, and 23.80±1.08 days, respectively). (d) Tumor size of the MCDmp group was smaller than that of the control group (arrow).
Fig. 2
Fig. 2
MCDmp-induced alterations in energy-dependent signaling pathways. (a) 18F-FDG PET scan shows decreased 18F-FDG uptake within tumors in the MCDmp group. T, tumor, b, urinary bladder. (b) The expression of the key enzymes for glycolysis (HK2 and PKM2) decreased significantly in the MCDmp group. (c) NAD+/NADH ratio was significantly increased (>3-fold) in melanoma tissues removed from the MCDmp group compared with the control group (*P=0.044, n=15 in each group). (d) Western-blot analysis showed activation of the LKB1/SIRT1/AMPK loop in the MCDmp group. There was no significant alteration in Nampt expression. Therefore, the NAD+/NADH ratio could be increased in the MCDmp group, independent of Nampt expression. In addition, the MCDmp intervention suppressed NF-κB p65 expression in the nuclear fraction of cells along with increased cytosolic IkB.
Fig. 3
Fig. 3
Tumor-suppressive role of SIRT1 under an energy restriction condition. (a) MTT assay of B16F10 melanoma cells cultured in a standard medium (SM, 11 mmol/l of glucose in RPMI-1640 medium) showed no significant cell proliferation at 24 or 48 h after treatment of SA3. However, cell proliferation was significantly suppressed after SA3 treatment when melanoma cells were culture in a low-glucose medium (LGM, 5.5 mmol/l of glucose). (b) SIRT1 and p-AMPK were increased in melanoma cells cultured in an LGM. However, the acetyl-p53 level was not significantly decreased despite an increased SIRT1 level. NF-κB p65 in the nuclear fraction was decreased under a low-glucose condition. (c) NF-κB level was decreased after SA3 treatment under both standard and low-glucose conditions.
Fig. 4
Fig. 4
Epigenetic control of the MCDmp intervention. (a) ChIP-PCR assay of the Pten and FoxO3a using the anti-H3K4me3 antibody confirmed increased H3K4 methylation after the MCDmp intervention. The numbers indicate the _target/input DNA ratios. (b) Western-blot analysis showed increased PTEN and FOXO3a as well as downstream _target proteins, Bim and Bax, in the MCDmp group. (c) The low-glucose condition without AMPK-activating agents also induced increased expression of PTEN, FOXO3a, and downstream _target Bim.
Fig. 5
Fig. 5
Diet-induced melanoma growth-suppression mechanism. (a) cDNA microarray analysis using melanoma tissues removed from the control and the MCDmp groups showed significant alterations in gene expression. Red represents upregulated mRNAs and green represents downregulated mRNAs. (b) The melanoma-specific proliferation markers, MITF and TYRP1, were decreased in the MCDmp group along with inhibition of VEGF, activation of the endogenous angiogenesis inhibitor (PEDF), and downregulation of the DEK oncogene. (c) Confocal analysis confirmed significantly decreased TYRP1 expression in the MCDmp group (green; anti-TYRP1, blue; DAPI). Inset images represent gross cut-sections of paraffin-embedded tumor tissue blocks showing decreased pigmentation in the MCDmp group. (d) Compared with MCD intervention, the MCDmp group showed higher expression of p-AMPK with suppression of the 4-EBP1, S6K1, ERK1/2, and MITF.
Fig. 6
Fig. 6
Dietary effects on cancer prevention. (a) Numerous tumor nodules developed within the liver after transfection of the HrasG12V and shP53 genes (Group 1). MCDmp intervention started after DNA transfection did not produce significant tumor-suppression activity (Group 2). However, fewer tumor nodules (arrow) developed when MCDmp was started 2 weeks before DNA transfection (Group 3). Post-mortem liver tissues also showed numerous tumor nodules throughout the liver in Group 1 (b)b, but fewer tumor nodules in Group 3 (c). (d) Kaplan–Meier survival analysis showed significant survival benefit only in Group 3. The median survival time of Group 1, Group 2, and Group 3 was 36±0.77, 36±0.45, and 45±1.20 days, respectively.
Fig. 7
Fig. 7
MCDmp-induced chemoprevention mechanism. (a) cDNA microarray analysis of the tumor-free liver tissues showed that numerous genes regulating glycolysis, tumorigenesis, and inflammation were downregulated in Group 3. (b) NF-κB and IL-6 expression was markedly decreased in Group 3 on real-time PCR. (c) H&E staining (×100, upper panel) of the tumor-free liver tissues obtained from Group 1 mice at 4 weeks after transfection shows a distorted lobular arrangement, but liver architecture is grossly preserved in Group 3. TRITC–phalloidin immunostaining (lower panel) shows severe dilatation and distortion of the bile canaliculi (arrows) in Group 1. However, the polyhedral architecture of the bile canaliculi is grossly preserved in Group 3. Scale bar=5 μm.
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